Recombinant AAV vectors are becoming an increasingly important gene transfer tool. Recent successes in clinical therapeutic applications suggest that the number of patients treated with these vectors is likely to increase rapidly in coming years. Preclinical studies have overwhelmingly supported the safety of rAAV gene therapy in numerous different tissues and animal models. However, a small number of studies have reported an excess incidence of hepatocellular carcinomas associated with rAAV treatment in mice, and in one case, this could be directly linked to vector DNA integration in a specific chromosomal locus. Other studies have found little evidence of such a link, despite exhaustive characterization of vector integration by massive parallel sequencing of DNA from tumors in rAAV infected mice. These inconsistent results leave the issue of rAAV genotoxicity in question, making it difficult to anticipate and disclose potential risks to patient groups. We have adopted a different strategy for the investigation of rAAV genotoxicity, essentially creating conditions where vector integration is likely to lead to tumor promotion in order to provide the sensitivity we need to detect oncogenic events, and then working back to dissect the mechanisms of genotoxicity as well as the vector elements that contribute to it. Using this strategy, we show in preliminary studies that we can readily measure an increase in tumor incidence associated with vector infection in a tumor-prone mouse strain (C3H/HeJ). This provides a valuable model for determining the comparative genotoxic potential of different rAAV vectors and, importantly, allows the characterization of the most likely mechanisms of oncogene activation. Using a rAAV vector designed to test the effects of strong read-through transcription on tumor promotion, our preliminary studies show tumor-associated vector genomes interacting with oncogenes through transcriptional read- through, transcriptional enhancer effects, and disruption of tumor suppressor genes. A second vector, of conventional design, was associated with excess tumors in this model through an apparently different mechanism, with little evidence for vector integration in tumors. Both of the vectors tested in our previous study were the self-complementary derivative of rAAV vector. In the proposed research, we will include three Aims designed to: 1. Directly compare self-complementary AAV and single-strand rAAV vectors for liver tumor promotion in the C3H/HeJ mouse model, and for differences in patterns of vector insertion in normal liver tissue from these animals. 2. Modify the scAAV vectors to determine what features contributed to tumor promotion, particularly from the vector that was not expected to activate oncogenes by transcriptional read- through. 3. Use deep sequencing technology to fully characterize rAAV integration sites in tumors and normal tissue with and without induction of cell-cycling. We will also relate the observed effects of vector infection in the tumor-prone mice to a tumor resistant mouse model to help extrapolate to liver tumor risk in humans. Together, these studies will greatly advance our understanding of the risk factors associated with rAAV vector gene therapy.